A fundamental dichotomy during multicellular differentiation of Dictyostelium discoideum establishes the prestalk/prespore pattern, an organization regulated by and sensitive to cell-cell communication and extracellular cAMP signal-response pathways. We have characterized the molecular functions and epistatic relationships of several genes that are critical for the formation of this pattern. Two cAMP receptors, CAR1 and CAR4, function antagonistically to one another for prespore/prestalk differentiation. This occurs, in part, through the differential regulation of glycogen synthase kinase 3 (GSK3) activity. Cells that lack CAR4 exhibit an enhanced expression of prespore markers and a corresponding increase in endogenous GSK3 activity, whereas developing cells that lack CAR1 or GSK3 fail to differentiate a prespore cell population. The RING/zipper protein rZIP also plays a central role in cell fate determination. Cells that lack rZIP have significantly increased expression of prespore genes and are inhibited in the transdifferentiation of prespore to prestalk cells. Conversely, overexpression of rZIP reduces prespore expression dramatically but promotes prestalk differentiation. Genetic analysis suggests a linkage of rZIP control and signalling through the cAMP receptors; biochemical analysis suggests that this linkage may be mediated through the activations or inhibitions of protein kinase A and MAP kinase, possibly by influencing homologous and heterologous protein interactions. Other potential downstream targets of receptor signalling include the brama-related chromatin protein DBR and the protein tyrosine phosphatase PTP4. As the cAMP receptors regulate development, they, in turn, are subject to developmental regulation. We have previously identified promoter elements and a nuclear factor that are essential for expression of CAR1. We have now purified this nuclear protein to near homogeneity and demonstrated by "footprint" analysis a zinc-dependent interaction with the nucleotide sequences that are essential for the regulated expression of CAR1 during early development.